Mutations in the gene encoding Parkin have been implicated in autosomal recessive juvenile parkinsonism (AR-JP). Parkin is a multi-domain protein that encodes a ubiquitin protein (E3) ligase, which ubiquitinates substrates for proteasome-mediated degradation, and was reported to play a specific role in the degradation of unfolded proteins. Mutations in the genes encoding alpha-synuclein (a-Syn) and UCH-Li also contribute to Parkinson's disease (PD), and provide compelling evidence for a genetic basis for this disease. Remarkably, the ubiquitin pathway degrades a-Syn, while UCH-Li is a neuronal protease that cleaves ubiquitin precursors. Consequently, there is an intriguing link between these proteins (Parkin, a-Syn, and UCH-LI) and the ubiquitin/proteasome pathway. Parkin contains RiNG domains that bind ubiquitin-conjugating (E2) enzymes. However, it is not known why Parkin contains two such sequences. Parkin also contains an amino-terminal ubiquitin-like (UbL) domain that might interact with the 26S proteasome. If UbL domains provide a general mechanism for interacting with the 26S proteasome, it would predict novel proteolytic functions for many UbL-containing proteins, including Parkin. Alpha-synuclein displays a propensity for self-association and forms insoluble aggregates in neuronal cells. These precipitates may contribute to PD, and similar phenomena could underlie the biochemical defects in other neurodegenerative diseases. In this regard, it is significant that a-Syn is ubiquitinated by Parkin and degraded by the proteasome, suggesting a link between the formation of intracellular aggregates and proteasome-mediated degradation. We will use our expertise in the ubiquitin field to define the biological functions of Parkin, by addressing the following critical questions. i). A potential interaction between Parkin and the proteasome will be examined in vitro, and in human cells. ii). We will determine if the two RING domains in Parkin are redundant, or play unique roles. iii). We will determine if the E2 enzymes UbcH7 and UbcH8 bind Parkin simultaneously to regulate the assembly of substrate-linked multi-Ub chains. iv). Our long-term objective is to characterize the ubiquitination of a-Syn and other physiological substrates by Parkin.